Human Mat2A Uses an Ordered Kinetic Mechanism and Is Stabilized but Not Regulated by Mat2B

Abstract

Methionine adenosyltransferase (MAT) catalyzes the adenosine 5′-triphosphate (ATP) and l-methionine (l-Met) dependent formation of S-adenosyl-l-methionine (SAM), the principal methyl donor of most biological transmethylation reactions. We carried out in-depth kinetic studies to further understand its mechanism and interaction with a potential regulator, Mat2B. The initial velocity pattern and results of product inhibition by SAM, phosphate, and pyrophosphate, and dead-end inhibition by the l-Met analog cycloleucine (l-cLeu) suggest that Mat2A follows a strictly ordered kinetic mechanism where ATP binds before l-Met and with SAM released prior to random release of phosphate and pyrophosphate. Isothermal titration calorimetry (ITC) showed binding of ATP to Mat2A with a K d of 80 ± 30 μM, which is close to the K m(ATP) of 50 ± 10 μM. In contrast, l-Met or l-cLeu showed no binding to Mat2A in the absence of ATP; however, binding to l-cLeu was observed in the presence of ATP. The ITC results are fully consistent with the product and dead-inhibition results obtained. We also carried out kinetic studies in the presence of the physiological regulator Mat2B. Under conditions where all Mat2A is found in complex with Mat2B, no significant change in the kinetic parameters was observed despite confirmation of a very high binding affinity of Mat2A to Mat2B (K d of 6 ± 1 nM). Finally, we found that while Mat2A is unstable at low concentrations (<100 nM), rapidly losing activity at 37 °C, it retained full activity for at least 2 h when Mat2B was present at the known 2:1 Mat2A/Mat2B stoichiometry.

The potential and challenges of targeting MTAP-negative cancers beyond synthetic lethality

“…MAT2B has been suggested to regulate MAT2A in low methionine or high methionine conditions by respectively activating or inhibiting MAT2A activity ( 95 ). Other data have suggested that the presence of MAT2B does not affect MAT2A activity but does improve MAT2A stability and longevity in low substrate concentrations ( 98 ). Therefore, these data suggest that the capacity of MAT2A inhibition via PF-9366 may be dependent on MAT2B levels or methionine/ATP availability.…”

Overview of Methionine Adenosyltransferase 2A (MAT2A) as an Anticancer Target: Structure, Function, and Inhibitors

“…Although many previous studies have shown that MAT2B can regulate MAT2A activity by changing its substrate affinity and sensitivity to product inhibition, 17−20 some studies have also demonstrated that the binding of MAT2B to MAT2A has no significant effect on the catalytic activity of MAT2A or physiologically relevant inhibition of SAM, meaning that the regulation of MAT2A activity by MAT2B does not appear to be direct and likely occurs through an unknown mechanism. 21 In addition, MAT2B interacts with many proteins that affect cell growth and signaling pathways. 22−28…”

Cysteine restriction induces ferroptosis depending on the polyamine biosynthetic pathway in hepatic cancer cells

“…MAT1 is a tetramer of MAT1A whereas MAT3 is a dimer of MAT1A (16, 17). MAT2 is composed of the catalytic subunit MAT2A and the regulatory subunit MAT2B that stabilizes MAT2A (16–18). MAT1 and MAT3 are expressed in the liver whereas MAT2 is expressed in almost all tissues of the body, developing liver and hepatocellular carcinoma but not in the normal liver (15, 19).…”